Animal tissues contain traces of materials, such as protoporphyrin, which fluoresce at a wavelength of 690 nm when excited by visible light. Such fluorescence is described, for example, in the article by R. H. Pottier et al., "Non-Invasive Technique for Obtaining Fluorescence Excitation and Emission Spectra In Vivo," Photochemistry and Photobiology, Vol. 44 pp. 679-687 (1986). Tissue fluorescence is also discussed in the article by William R. Potter and Thomas S. Mang, "Photofrin II Levels By In Vivo Photometry," Progress in Clinical and Biological Research, Vol. 170 pp. 177-186 (1984). The above articles are incorporated herein by reference.
The fluorescent tumor localizing photosensitizer Photofrin II is retained by abnormal tissue such as tumors at a higher level than most surrounding normal tissues, and therefore it is diagnostically useful to supply Photofrin II to the tissues, and then to illuminate the tissue with light to detect by the fluorescent response whether abnormal tissue is present.
In the therapeutic use of this material (referred to as photodynamic therapy, or PDT), large doses of 630 nm light are used both to activate the fluorescence of the sensitizer (such as Photofrin II) and to selectively destroy the tumor by a photochemical reaction However, the fluorescent response of tissues may be created by excitation using incident light with wavelengths in the 600 nm region, which is in the visible spectrum, and thus there is a problem with stray light causing fluorescence which may be interpreted as arising from abnormal tissue. Thus, there is a need for a system which can accurately differentiate between fluorescence arising from sensitizer in normal tissue and that arising from sensitizer in abnormal tissue, especially in vivo. In addition, there is a need for distinguishing between fluorescence arising from low levels of fluorescent tumor localizers (i.e., sensitizers such as Photofrin II) and natural tissue background fluorescence.
There is especially a need for a fluorometer which can detect abnormal cells which are within a mass of tissue, such as within a group of lymph nodes, without the need for slicing the tissue open and inspecting each sliced segment in a superficial manner, as has been done in the past. Thus, it is an object of this invention to provide a method and apparatus of fluorometry with the capability of effectively penetrating a mass of tissue for purposes of detecting abnormal tissue.
One characteristic of presently used PDT methods is the need to use therapeutic levels of the sensitizer which result in highly photosensitive skin for long periods of time, often on the order of four to six weeks. This skin sensitivity requires the patient to remain indoors during daylight hours after injection until the photosensitivity has decreased.
Thus, long and high photosensitivity is a significant disadvantage to the use of this drug for detection or localization. The need to use high levels of the drug is a result of the natural background fluorescence of the tissue, which tends to vary in a random fashion from point to point.
In one system, an imaging device uses 400 nm absorption for superficial excitation of bladder tissue. H. Baumgartner et al., "A Fluorescent Imaging Device for Endoscopic Detection of Early Stage Cancer--Instrumental and Experimental Studies," Photochemistry and Photobiology, Vol. 46, No. 5, pp. 759-763 (1987). In this system, tissue is first scanned using light in the violet region of the spectrum, and a subsequent scan with green or blue light from an argon laser is used to excite the tissue background and subtract this contribution to the image. There are certain disadvantages to this approach, however, one of which is that the tissue excitation by the two wavelengths is done in an alternating fashion, such that real-time images of in vivo tissues are not achievable, since registration of the image would have to be maintained for the two excitation wavelengths. Furthermore, it would be impractical to use this type of imaging with light in the 600 nm range because scattering of the light by tissue would cause resolution to be very poor.
However, imaging with wavelengths of light in the 600 nm range is highly desirable because of the deep penetration of such wavelengths. There is therefore a need for a system for in vivo fluorometry which produces real-time images which may utilize longer wavelengths for noninvasive examination of tissue to the maximum depth possible, especially for use with handheld probes. There is also a need for a system which utilizes relatively low levels of sensitizing chemicals such as Photofrin II, so as to greatly reduce or eliminate clinically significant photosensitivity.
It is an object of this invention to provide a method and apparatus for in vivo fluorometry which can be implemented in a handheld nonimaging probe where sequential tissue excitation is not feasible.